MOLECULAR IDENTIFICATION OF MULTIFORM PEPTIDES RELATED TO PROTEINASE INHIBITOR FROM JAPANESE TOAD (Bufo bufo formosus)

Yang, X.(1), Kobayashi, S.(1), Rádis-Baptista, G.(2), Yamane, T.(2), Kubo, T.(1) 

(1)National Institute of Advanced Industrial Science and Technology, Neurobiology Research Institute, Molecular Neurophysiology, Tsukuba, Japan (2)Institute Butantan, Molecular Toxinology Laboratory, SãoPaulo, Brazil

Frog and toad skins are well known to be a rich source of bioactive peptides. It is also recognized that many of the amphibian skin peptides have counterparts in the gastrointestinal tract and the nervous system of higher vertebrates. Recently, there is a growing interest in therapeutic and industrial applications of antimicrobial and others bioactive peptides from the skin secretions. “Gama ointment” made from skin secretion of Japanese toads is one of the traditional medicines to treat wound, injury and infectious skin diseases.  Heart stimulation, local anesthesia and diuretic effects are also observed in extract of toad skin secretions. To identify the biologically active substances we prepared a cDNA library from dorsal skin of the toad (Bufo bufo formosus), and screened the library using primers degenerated from the core sequence “CA(N/D)”, which is conserved among some of the bioactive peptides, such as chemokine family. Among the identified cDNAs we focused on the clones coding a novel and characteristic cysteine framework. Four clones so far identified show common structural features: a signal peptide sequence followed by one to five repetitive domain(s). Each of the domains contains strictly spaced eight cysteine residues but the overall degree of sequence similarity is low.  This characteristic structure is also found in whey acidic protein, elafin (elastase inhibitor), chelonianin (trypsin and subtilisin inhibitor), WDNM1 protein (involved in the metastasis), Kallmann syndrome protein, caltrin-like protein II (inhibits calcium transport). Biochemical analysis of recombinant polypeptides from the clones is under investigation.

CORRESPONDENCE TO:

Tai Kubo, 1-1-1 Higashi, AIST Tsukuba Central 6, 305-8566, Japan, Email: tai.kubo@aist.go.jp